Mapping a Systematic Ribozyme Fitness Landscape Reveals a Frustrated Evolutionary Network for Self-Aminoacylating RNA

Abstract

Molecular evolution can be conceptualized as a walk over a "fitness landscape", or the function of fitness (e.g., catalytic activity) over the space of all possible sequences. Understanding evolution requires knowing the structure of the fitness landscape and identifying the viable evolutionary pathways through the landscape. However, the fitness landscape for any catalytic biomolecule is largely unknown. The evolution of catalytic RNA is of special interest because RNA is believed to have been foundational to early life. In particular, an essential activity leading to the genetic code would be the reaction of ribozymes with activated amino acids, such as 5(4 H)-oxazolones, to form aminoacyl-RNA. Here we combine in vitro selection with a massively parallel kinetic assay to map a fitness landscape for self-aminoacylating RNA, with nearly complete coverage of sequence space in a central 21-nucleotide region. The method (SCAPE: sequencing to measure catalytic activity paired with in vitro evolution) shows that the landscape contains three major ribozyme families (landscape peaks). An analysis of evolutionary pathways shows that, while local optimization within a ribozyme family would be possible, optimization of activity over the entire landscape would be frustrated by large valleys of low activity. The sequence motifs associated with each peak represent different solutions to the problem of catalysis, so the inability to traverse the landscape globally corresponds to an inability to restructure the ribozyme without losing activity. The frustrated nature of the evolutionary network suggests that chance emergence of a ribozyme motif would be more important than optimization by natural selection.

Figure 1

In vitro selection for aminoacylation ribozymes. (A) Selection began with DNA templates containing a transcription promoter (gray) and a central region of 21 random-sequence residues (red or blue) flanked by constant regions (black). These templates were transcribed into RNA and incubated with BYO. Aminoacylated RNAs (red) were isolated using streptavidin beads and amplified by RT-PCR for the next round of selection. (B) Pool composition over Rounds 4–6 after clustering. The top 20 families are indicated in non-neutral colors; gray corresponds to unclustered sequences; white corresponds to families with rank by abundance >20. Multiple families from submotif 1A (purple), 1B (dark blue), 1C (cyan), Motif 2 (green), and Motif 3 (yellow) are shown. Inset: Abundance of the top 20 families in Rounds 4–6 (same color scheme, except that the dotted black line corresponds to families of rank >20). (C) SeqLogo representations of the motifs.

Figure 2

Emergence of ribozymes and kinetic characteristics. (A) In k-Seq, an RNA pool enriched for active ribozymes is reacted at multiple BYO concentrations, in triplicate. Captured RNA is then reverse-transcribed and sequenced. Activity curves are constructed for sequences detected in the enriched pool. (B) Aminoacylation at various [BYO] for ribozyme S-2.1-a observed by both gel shift and k-Seq Data for all other measured ribozymes are shown in Supporting Figure S3. Error bars correspond to standard deviation among triplicates. (C) Correlation between catalytic enhancement of ten ribozymes, measured by gel shift assay and k-Seq Error bars correspond to standard deviation among triplicates (k-Seq) or 2–3 replicates (gel assay) (R² = 0.87; Supporting Table S1). Dotted orange line indicates line of unity.

Figure 3

Aminoacylation site and landscape ruggedness. The likely site of BYO modification on ribozyme S-1A.1-a was identified by stalling of reverse transcription, resulting in a truncated product (A). The site, G65, was verified by loss of activity upon 2′-O-methylation, assayed by streptavidin gel shift after BYO reaction (B). 2′-O-Methylation of an adjacent site (C64) did not show loss of activity. (C) Average correlation of fitness effects γ_d as a function of edit distance d, shown for the sequence families around the five most abundant centers: 2.1 (magenta), 1A.1 (red), 1B.1 (orange), 1B.2 (green), 1A.2 (blue).

Figure 4

Evolutionary pathways for aminoacylation ribozymes. (A) Catalytic enhancement along a best pathway discovered from the center of Family 1B.1 (pink, S-1B.1-a), to 1A.1 (purple, S-1A.1-a), to 2.1 (cyan, S-2.1-a), to 2.2 (gray, S-2.2-a). Capital letters denote sequence positions changing at each step; underscore indicates a deletion. A large drop in activity is required for several mutations between Motif 1 and Motif 2. Error bars are standard deviation from triplicate measurements (only top bar is shown). Also see Supporting Figure S11. Asterisk (*) indicates a sequence that was found in only one replicate (RS1). (B) Evolutionary network displaying the 10 best pathways discovered between the centers of six key families (1A.1, 1B.1, 1C.1, 2.1, 2.2, and 3.1) representing each motif and submotif and the two most active centers from Motif 2. Each node is an individual sequence with activity measured by k-Seq indicated by color (see legend; red indicates activity at or below the baseline rate). The lines indicate mutational distance between sequences (solid black line = 1 mutation). Dotted lines indicate sequences at baseline activity (see legend). The majority (67%) of the edits along these pathways are substitutions; the remainder are indels.